DE60025758T2 - ANALYSIS DEVICE AND ITS USE - Google Patents

Abstract

A test device and a kit for conducting an assay for the determination of an analyte in a sample comprises (i) a housing (1, 2), and within said housing, (ii) a flow matrix (6) allowing liquid to be transported by capillary action and having at least one zone with immobilized capturing agent capable of directly or indirectly binding to the analyte, (iii) a liquid container (13) for sample liquid, and (iv) at least one liquid container for liquid other than sample liquid. The device further comprises (v) separation means (5) between the flow matrix (6) and the liquid containers (13), wherein said separation means (5) are mounted in a movable relationship with the liquid containers to in a first position prevent liquid contact of the flow matrix (6) with the liquid containers (13), and in a second position permit liquid receiving contact of the flow matrix (6) with the liquid containers (13). An assay method for determining an analyte in a sample wherein the sample and assay liquids are flown through a flow matrix to reach the reaction zone in a predetermined sequence uses the device for carrying out the method. The use of the device comprises testing for an allergy, inflammation or autoimmune disease.

Description

Field of the invention

The The present invention relates to a novel solid phase analysis device to carry out Analyzes, in particular immunochromatographic analyzes, for determination of analytes in samples and methods of using the device.

Background of the invention

A Type of solid phase analysis devices comprise a plate-shaped flow matrix from absorbent Material, usually a membrane strip, e.g. made of cellulose nitrate or fiberglass, in the liquid lateral (i.e., in the plane of the strip) by capillary forces in the Membrane can be transported. The membrane usually has a sample application zone and a destination zone downstream of the sample application zone. In the destination zone is common a trapping agent for immobilized the analyte. To perform an analysis will the application zone with the liquid sample of analyte of interest to be analyzed brought into contact. The device is kept under conditions sufficient to provide a liquid capillary action to transport the analyte of interest when in the sample is present, through the membrane strip to the destination zone, where the analyte is captured. The capillary liquid flow becomes common by an absorption pad or the like at the downstream end of the strip. A determination reagent, usually labeled, is then added upstream of the detection zone and enters with the captured analyte in the detection zone in response, and the Amount of captured analyte is measured. The determining reagent is often previously deposited in or on the membrane strip, e.g. in shape of diffusely moving particles, the fluorophore or chromogenic Groups, either upstream of the sample application zone or between the sample application zone and the destination zone.

EP-A-306 336 describes an analysis device of the type described above general type, wherein the strip of absorbent material enclosed in a housing is. The case has a first opening for the introduction the sample into the device, and a second opening for introducing a different from the sample liquid reagent into the device, e.g. of a component of the used a signal generating system. The device can except the two openings additional Facilities for introducing additional analysis reagents into the device, e.g. a third opening in the housing or a in the device included breakable container with liquid Reagent. The device should also, if the operator all Stages in quick succession, allow precise reagent additions.

WHERE 99/36776 describes an analytical method of the one described above general style based on the finding that a zonal Hike desired analysis liquids can be obtained in a specific order when the liquids simultaneously or almost simultaneously adjacent zones in strips be added.

A Analysis Implementation with one of those described in the two aforementioned publications However, devices require that the operator have a number of Stages in a short time, and in particular the simultaneous addition of liquids by the method WO 99/36776 may be for be difficult or impractical to carry out the operator.

U.S. 4,959,324 . US 5 260 221 and EP 0 427 534 describe test devices having two bibulous strips containing buffer and destination reagent separated by a gap bridged by a movable element.

Summary of the invention

task The present invention is the provision of an analysis device for execution an analysis for determining an analyte, wherein the device the simultaneous initiation of a flow of the sample and of at least another analysis fluid allows. A Another object of the present invention is the provision an analytical device which is suitable for carrying out a Sequential analysis with a specific flow (flow) of the sample and the analysis fluids through the device.

A Another object of the present invention is the provision a device designed for the operator is easy to operate and a minimum of process steps requires.

To The present invention addresses the above and other objects and advantages with a test device for performing a Analysis for the determination of an analyte in a sample, as described in appended claim 1 is achieved achieved.

The term "liquid container" is to be interpreted broadly and basically includes either Liquid-retaining element or device capable of absorbing and dispensing liquid. The liquid container may therefore be a receptacle or a recess the opening of which is executed to the flow matrix, and wherein the opening is closed or sealed by the above-mentioned liquid-tight member. The liquid container may also be a body that can absorb and retain a certain amount of an aqueous liquid, such as a pillow or a spongy body. Usually, such a body is located in a depression or other space which is closed by the liquid-tight element.

In an embodiment the liquid containers are adjacent to a surface, usually the upper surface, the flow matrix attached, and the separating means has a between the liquid containers and the flow matrix sandwiched flat liquid-tight element. Preferably, this is liquid-tight Element at least partially removable from the housing and may e.g. be a pull-out movie.

In an alternative embodiment are the liquid containers in Movable relative to the flow matrix, that is, in a first position, the liquid containers of the flow matrix separated and can be placed in a second position, wherein the container in a liquid-transferring contact with the flow matrix stand.

Short description of drawings

1 is a perspective view of an embodiment of a device according to the invention.

2 is a plan view of the lower part of the device in 1 ,

3 is a partially transparent view from below of the upper part of the device in FIG 1 ,

4 is a side elevation of the device in FIG 1 ,

5 is an exploded view that is the side elevation in 4 equivalent.

6 is an exploded partial view of another embodiment of the device according to the invention.

Detailed description the invention

As this is best in 1 is shown in the 1 to 5 illustrated device an upper housing part 1 and a lower housing part 2 of a material which is inert with respect to the sample and any analyte reagent to be carried out with the device, eg polystyrene or polypropylene. The upper housing part has a sample well opening 3 (here conical) and a determination window 4 on. In 1 also becomes a removable release agent to be described below 5 shown.

With reference in particular to the 2 but also the 3 to 5 has the lower housing part 2 a test strip of absorbent material mounted therein (which is a porous material capable of conveying an aqueous medium longitudinally due to capillary action), eg, nitrocellulose on a polyester pad. To avoid capillary action along the edges of the strip, the strip is 6 attached to a rib (dashed line) at the bottom of the housing part, wherein the rib is narrower than the width of the strip. The positioning of the strip 6 in the housing is made by guide elements 7 facilitated. Near the upstream end of the strip 6 (left in 2 ) is a filter piece 8th containing a diffusely mobile determination reagent. Such a determining reagent may be, for example, a conjugate between a marking particle and a reactant capable of binding to the analyte. Further downstream and below and within the destination window 4 attached is a reaction zone 9 on the strip containing an immobilized reactant capable of binding an analyte to be determined. In the case shown is also a calibrator zone 10 present, which contains a certain amount of an immobilized calibrator substance, eg an analyte. At the membrane strip 6 also shown is a flow barrier 11 , here specifically a piece of a film element that the filter piece 8th covered and against the opening 3 in the housing part 1 runs. The function of the flow barrier 11 will be described later.

After the 3 to 5 contains the upper housing part 1 at the upstream end of the membrane strip 6 an edition 12 of a liquid-absorbent material to serve as a container for fluid or buffer. The opening 3 in the housing part 1 ( 1 ) is used to introduce sample to the membrane 6 , In the case shown is below the opening 3 a filter element 13 (which may optionally consist of two or more separate filters) for analyzes in which the sample liquid must be filtered, for example, if the sample is whole blood, and blood cells are to be separated. The puff pillow 12 thus forms a buffer liquid container, hereinafter referred to as buffer pad, and through the sample port 3 and the filter element 13 defined space forms a sample well or a sample container.

At the downstream end of the membrane strip 6 is a pillow 14 of absorbent material, the purpose of which is to maintain a capillary flow of the analysis fluids through the membrane strip 6 to support.

The above-mentioned separating element 5 , here a liquid-tight pull-out film, is at the upstream part of the membrane strip 6 attached to contact between the membrane strip 6 and the subparts of the buffer pad 12 or the sample filter 13 to prevent. The film 5 is arranged so that it can be manually removed by pulling it away from the device and thereby the upper surface of the membrane strip 6 to the buffer cushion 12 (except for the part of the membrane strip passing through the flow barrier film 11 is covered) or the sample filter 13 suspend so that the membrane strip 6 in simultaneous or near simultaneous fluid-receiving contact with the buffer pad 12 and the filter 13 is brought into the sample well. The upper housing part 1 has a recess 15 for the buffer cushion 12 which is designed to hold the pillow against the pull-out film 5 presses, and thus against the membrane strip 6 and the flow film 11 if the pull-out film 5 Will get removed. To a liquid-tight enclosure of the pad 12 in the depression 15 ensure the pull-out film against the edges of the recess 15 tightly sealed, eg by welding. Although in the above case, the pull-out film 5 It can be completely removed from the device, it is of course sufficient that the film 5 from the membrane strip 6 is pulled away to such an extent that the candidate membrane strip surface portions are exposed to the sample or buffer liquids.

A Analysis for an analyte in the sample can be made with the above described device are carried out as follows.

The device is usually provided ready for use, with the buffer pad 12 impregnated with buffer solution (flow liquid) and the determining reagent in the filter 8th precursor and the appropriate suitable capture reagents in the reaction (or destination zone) 9 or the calibration zone 10 are immobilized. For example, if the analyte to be tested is an antigen, the determining reagent may be in the filter 8th for example, an antibody to the antigen coupled to a fluorogen-labeled particle, the immobilized reactant in the reaction zone 9 may be an antibody to the antigen, and the calibrator in the calibration zone 10 may be the analyte or an analyte-analog.

A certain amount of the sample is through the opening 3 in the housing part 1 added. All the necessary analysis fluids, ie sample fluid and buffer fluid in this case, are then present in the device, the pull-out film 5 however, effectively prevents contact between the respective liquids and the membrane strip 6 , The analysis is then done by the operator by removing the pull-out film 5 started, causing the membrane strip 6 at the same time liquid-receiving contact with the buffer pad 12 and the sample liquid in the sample well 3 is brought.

Buffer fluid from the pillow 12 penetrates into the membrane strip 6 via its remote upstream end portion, which is in direct contact with the pillow 12 stands (see 5 ), and will be on the membrane strip 6 transported downstream by capillary force. At the same time sample liquid penetrates into the membrane strip 6 and is transported in the downstream direction of the strip. There is thus a flow of the sample liquid, followed directly by a flow pulse of the buffer liquid. The determination reagent filter 8th and much of the buffer pad 12 are however from the membrane strip 6 through the flow barrier film 11 separated. Buffer liquid contained in the membrane strip 6 is transported, will penetrate and through the filter 8th transports and carries the precipitating reagent deposited here to form a determining reagent flow impulse. This determination reagent flow pulse follows in order of the sample flow and the buffer flow pulse. Buffer in the membrane strip 6 is transported after the determination reagent from the filter 8th is removed forms a second buffer flow pulse following the determination reagent flow pulse.

The above-mentioned various liquid flow rates are along the membrane strip 6 transported in the order given, ie, sample flow, first buffer flow, destination reagent flow and second buffer flow, and optionally the calibrator zone 10 and the reaction zone 9 to reach. In the reaction zone 9 analyte present in the sample is captured by the reagent immobilized in the membrane. The resulting analyte / capture reagent complex is washed by the subsequent first buffer flow, and the analyte / reagent complex is then reacted with the assay reagent contained in the assay reagent flow to form a detectable assay reagent / capture reagent complex. The latter is finally washed by the second buffer flow. In the calibration zone 10 the particular amount of analyte contained therein will react with the determining reagent in the determining reagent flow to form a determinable determination reagent / analyte complex. The fluid from the buffer pad 12 will consequently dissolve washing, determining reagent and washing. By measuring over in the opening 4 in the housing part 1 formed determination windows may be from the signal intensity of that in the reaction zone 9 capture reagent and correlate with that in the calibration zone 10 The amount of analyte in the sample can be determined.

Out the above statements It can be seen that an analysis with the described device easy and convenient to perform is, and a simultaneous initiation of different analysis liquid streams allows. Once the sample has been placed in the sample well, the extractable Movie to be removed. The liquid in The buffer pad and the sample are thereby in contact with the Brought membrane strips, and the desired sequential transport different liquid streams begins.

6 illustrates a different embodiment of the device according to the invention. This device is different from the one in the 2 to 5 in that (i) the only buffer pad 12 in the embodiment of the 2 to 5 through three different buffer cushions 12'a . 12'b and 12'c (ii) the flow barrier film 11 is omitted and the determination reagent filter, here with 8th' referred to below the central buffer pad 12'b is arranged. This embodiment also gives the same sequence of liquid flows as in FIGS 2 to 5 as soon as the pull-out film 5 ie, sample stream, first wash stream, destination reagent stream and second wash stream.

In another modification of the device according to the invention are the buffer pads 12'a and 12'b in 6 combined into a single buffer pad, and the assay reagent filter 8th' in 6 (with or without flow barrier film, similar to the barrier film 11 in 5 ) is located below the downstream portion of the combined buffer pad. This embodiment also gives the same sequence of liquid flows after removal of the pull-out film. The buffer cushion 12'c washes, and the combined cushion pad 12'a . 12'b dissolve the determination reagent and then wash.

In a further modification of the device according to the invention, the cushion pad arrangement of 6 three separate buffer pads 12'a . 12'b and 12'c on, but the destination reagent filter 8th in the 2 to 5 is removed and the determining reagent is in the central buffer pad 12'b stored in stock. Optionally, one or both of the two flanking cushion pads may be used 12'a and 12'b be omitted in this embodiment.

Other Buffer pad arrangements as well as other variations and changes the device described above by way of example only, are for a person skilled in the art recognizable.

In is the reaction (or destination) zone described above immobilized a reactant capable of specific binding of the analyte (via covalent bonding via physical adsorption, over biospecific affinity, via immobilized Particles to which the reactant is covalently bound, etc.). It can but also an agent that can react with the reactant in The membrane may be immobilized and the reactant may then be together be added with the sample or in the membrane in one area or a zone upstream of the reaction zone. Such agent may be an element of a specific binding pair (spb), and the reactant is then transferred to the other element of the spb coupled or conjugated. Exemplary specific binding pairs include immunological binding pairs, e.g. Antigen antibodies and Hapten antibody, Biotin-avidin or Streptavidin, lectin-sugar, hormone-hormone receptor, nucleic acid duplex. The reaction zone may e.g. have streptavidin immobilized therein, and the capture reagent for the analyte may be biotinylated.

On similar The calibration zone may be a binder for the calibrator substance and not the calibrator substance per se. The binder is usually an element of a specific binding pair, e.g. one of the mentioned above, while the other element of the specific binding pair to the calibrator substance coupled or conjugated, which is added back to the sample may already be deposited or upstream of the calibration zone can be. Streptavidin may e.g. immobilized in the calibrator zone be while the calibrator substance is biotinylated.

For further Details of analytical devices of the type discussed here and in particular with regard to flow matrices, sequence analyzes, Calibrator systems and reagents are described e.g. on our published international applications WO 99/36776, WO 99/36777 and WO 99/36780.

The analytes to be determined using the present device will be readily apparent to one skilled in the art. Usually, however, the analyte is a biospecific Affinity reactant, eg an antibody, or other protein, hapten, nucleic acid or polynucleotide, such as a DNA sequence. In the latter case, the reaction zone may contain streptavidin, and the DNA sequence to which the analyte sequence is to be hybridized may be biotinylated.

The present device allows a suitable pretreatment the sample before starting the analysis.

The The present device can also be used to perform analyzes in our PCT application WO 99/60402 be described type, wherein the flow matrix upstream of the reaction (destination) zone contains a chromatographic separation zone to sample components that otherwise disturb the determination of the analyte or influence separate.

Claims (23)

A test device for carrying out an assay for determining an analyte in a sample, the device comprising a housing ( 1 . 2 ), i) has a flow matrix ( 6 ) in the form of a membrane strip made of an absorbent material located inside the housing, wherein it contains the material in the flow matrix ( 6 ) the liquid allows to be transported by capillary action, the flow matrix has at least one zone with an immobilized capture agent that can bind directly or indirectly to the analyte, ii) a sample well opening ( 3 ) for sample liquid and iii) at least one liquid container ( 12 ) for a liquid other than the sample liquid, characterized in that the sample well opening ( 3 ) for the sample liquid and the liquid container (s) ( 12 ) for liquid other than the sample liquid above the membrane strip ( 6 ) are arranged, and that a separating element ( 5 ) is provided to check the contact between the test strip ( 6 ) and the bottom parts of the container (s) ( 12 ) or the trough ( 3 ), the separating element ( 5 ) is manually removable so that it essentially simultaneously the top of the test strip ( 6 ) opposite the bottom parts of the sample well ( 3 ) or the liquid container ( 12 ). Test device according to claim 1, characterized in that the flow matrix ( 6 ) is flat and the fluid flow within the matrix is lateral. Test device according to claim 2, characterized in that the liquid container ( 12 ) and the trough ( 3 ) adjacent to an area of the flow matrix ( 6 ), and the release agent is a flat liquid-tight element ( 5 ), which between the liquid containers ( 12 ) or the sample well ( 3 ) and the flow matrix ( 6 ) is sandwiched. Test device according to one of claims 2 to 3, characterized in that the liquid-tight element ( 5 ) at least partially from the housing ( 1 . 2 ) is removable. Test device according to claim 4, characterized in that the liquid-tight element ( 5 ) is a pull-out element, eg a pull-out film or film. Test device according to one of claims 1 to 5, characterized in that the at least one liquid container for a different liquid from the sample liquid at least one container with flow liquid ( 12 ), such as a buffer solution. Test device according to claim 6, characterized in that the liquid container or the liquid container for the flow liquid in the form of an absorbent pad or sponge ( 12 ) are present. Test device according to one of claims 1 to 7, characterized in that the at least one liquid container for from the sample liquid different liquid a container for an analytic having detectable reagent. Test device according to claim 8, characterized in that that the liquid container for the analytic determinable reagent in the form of an absorbent pad or a Sponge is present. Test device according to claim 8 or 9, characterized characterized in that at least one liquid container for flow fluid upstream and / or downstream from the container is provided with the analytically determinable reagent. Test device according to one of claims 1 to 7, characterized in that the flow matrix ( 6 ) has a zone which contains the analytically determinable reagent in advance in the matrix or in an element present on the matrix ( 8th ) having. Test device according to claim 11, characterized in that that a first container for flow fluid above and along the zone with the analytically determinable reagent is provided. Test device according to claim 12, characterized in that that at least a second container for flow fluid upstream from the first container is provided and / or at least a third container downstream of first container is provided. Test device according to claim 11, characterized in that the first container ( 12 ) for flow fluid upstream of and at least partially above and along the zone of analyte-determinable reagent. Test device according to claim 11, characterized in that that at least a second container for flow fluid downstream from the first container is provided. Test device according to claim 14 or 15, characterized in that a separating element ( 11 ) above the zone ( 8th ) with analytically determinable reagent to allow direct contact between the first container ( 12 ) for flow liquid and the zone with the analytically determinable reagent, if the separating element ( 5 ) in the second position. Test device according to one of claims 1 to 16, characterized in that in the flow matrix ( 6 ) immobilized capture agent is part of a specific binding pair and that the other part of the specific binding pair is part of or attached to the reagent capable of binding the analyte. Test device according to claim 17, characterized in that that the specific binding pair is antigen-antibody, hapten-antibody, biotin-avidin, biotin-streptavidin or a nucleic acid duplex is. Test device according to one of claims 8 to 18, characterized in that the analytically determinable reagent is marked as e.g. by a fluorophore or a chromophore. Method for performing an assay for determination an analyte in a sample, the method flowing through it of sample and assay fluids through a flow matrix includes a reaction zone in this flow matrix in a specific Achieve sequence, characterized in that for carrying out the Method a device according to one of claims 1 to 19 is used. Use of a device according to one of claims 1 to 18 for testing an analyte in a sample that is a disease indicates selected from allergy, inflammation and autoimmune diseases. Use according to claim 21, wherein the analyte is a is specific immunoglobulin. Kit to carry out an assay method, wherein the kit is the device according to one of claims 1 to 19 in combination with another assay component (s).